Tyrphostin A9 inhibits the TNF-induced respiratory burst of human PMNs and TNF-induced tyrosine phosphorylation of pyk2 but not their bactericidal activity. (a) Inhibition of TNF-stimulated H₂O₂ release. PMNs were plated in FBS-coated 96-well plates and preincubated for 30 minutes with the indicated concentrations of tyrphostin A9. Cells were then unstimulated (–) or stimulated (+) either with TNF (100 ng/mL; gray bars) or PMA (100 ng/mL; filled bars), and H₂O₂ release was recorded 60 minutes later as mean ± SEM of triplicates. (b) Lack of inhibition of bacterial killing. PMNs were preincubated (30 minutes at 4°C) with 0 (filled squares) or 5 μM (open squares) tyrphostin A9 and then infected with 10% autologous serum-opsonized Salmonella typhimurium. Cells were lysed at indicated times and CFUs were determined. Survival of bacteria without PMNs is shown with 0 (filled circles) or 5 μM (open circles) tyrphostin A9. Mean ± SEM of triplicates; most error bars fall within the symbols. (c) PMNs were plated on FBS-coated plates, preincubated for 30 minutes in the presence or absence of tyrphostin A9 (2 μM), and then stimulated with TNF (250 ng/mL) or left untreated for 60 minutes, as indicated. Cell lysates were immunoprecipitated (IP) with anti-pyk2 antibody. Total cell lysate and immunoprecipitates were separated by reducing SDS-PAGE, transferred to nitrocellulose, and Western blotted (WB) with anti-phosphotyrosine mAb (top) or anti-pyk2 antibody (bottom) followed by ECL detection. Molecular mass markers are indicated in kilodaltons.